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Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72 h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.

Biofilm formation is an important virulence factor for pathogenic fungi. Both yeasts and filamentous fungi can adhere to biotic and abiotic surfaces, developing into highly organized communities that are resistant to antimicrobials and environmental conditions. In recent years, new genera of fungi have been correlated with biofilm formation. However, Candida biofilms remain the most widely studied from the morphological and molecular perspectives. Biofilms formed by yeast and filamentous fungi present differences, and studies of polymicrobial communities have become increasingly important. A key feature of resistance is the extracellular matrix, which covers and protects biofilm cells from the surrounding environment. Furthermore, to achieve cell–cell communication, microorganisms secrete quorum-sensing molecules that control their biological activities and behaviors and play a role in fungal resistance and pathogenicity. Several in vitro techniques have been developed to study fungal biofilms, from colorimetric methods to omics approaches that aim to identify new therapeutic strategies by developing new compounds to combat these microbial communities as well as new diagnostic tools to identify these complex formations in vivo. In this review, recent advances related to pathogenic fungal biofilms are addressed.

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Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment.

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Mammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting.

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